Heterogeneous glycosylation and methylation of the Aeromonas caviae flagellin

Abstract Bacterial swimming is mediated by the rotation of a flagellar filament. Many bacteria are now known to be able to O‐glycosylate their flagellins, the proteins that make up the flagellar filament. For bacteria that use nonulosonic acid sugars such as pseudaminic acid, this glycosylation process is essential for the formation of a functional flagellum. However, the specific role of glycosylation remains elusive. Aeromonas caviae is a model for this process as it has a genetically simple glycosylation system. Here, we investigated the localization of the glycans on the A. caviae flagellum filament. Using mass spectrometry it was revealed that pseudaminic acid O‐glycosylation was heterogeneous with no serine or threonine sites that were constantly glycosylated. Site‐directed mutagenesis of particular glycosylation sites in most cases resulted in strains that had reduced motility and produced less detectable flagellin on Western blots. For flagellin O‐linked glycosylation, there is no known consensus sequence, although hydrophobic amino acids have been suggested to play a role. We, therefore, performed site‐directed mutagenesis of isoleucine or leucine residues flanking the sites of glycosylation and demonstrated a reduction in motility and the amount of flagellin present in the cells, indicating a role for these hydrophobic amino acids in the flagellin glycosylation process.

An increasing number of bacteria including Aeromonas species have now been shown to be able to O-glycosylate their flagellins, linking sugars onto serine or threonine residues within the central D2/D3 domain of the protein, a region that is thought to form the surface of the flagellar filament (Yonekura et al., 2003). Many of these bacteria use nonulosonic acids such as pseudaminic acid or legionaminic acid or their derivatives (Schirm et al., 2005;Thibault et al., 2001). This glycosylation process is essential for motility and the formation of the flagellar filament and is controlled by the protein Maf (Parker et al., 2012. O-linked flagellin glycosylation has been characterized most extensively in Campylobacter species that possess complex glycosylation islands and glycosylate their flagellins with a variety of nonulosonate sugars (Schirm et al., 2005;Thibault et al., 2001). Studies in Campylobacter jejuni 81-176 revealed that 19 serine or threonine residues were modified by pseudaminic acid or derivatives, with all but one of these sites being present within the central D2/D3 domain of the protein (Thibault et al., 2001). Furthermore, intact mass spectrometry (MS) analysis of flagellins demonstrated different flagellin components to exist at one time, suggesting heterogeneity (Thibault et al., 2001). However, in C. jejuni 81-176 it was established that the identified sites are "usually" occupied, suggesting a small amount of heterogeneity with regard to the site of modification (Thibault et al., 2001).
Aeromonas caviae Sch3 attaches the basic form pseudaminic acid (Pse5Ac7Ac) to the central D2/D3 domain of the FlaA and FlaB polar flagellin proteins (Gray et al., 2019;Tabei et al., 2009). The exact sites of flagellin glycosylation have not been identified in A. caviae; however, how this bacterium modifies its flagellins may indicate the function of this sugar at the cell surface. For example, if glycosylation is homologous, by always being present on the same serine or threonine residues, it may have a structural role, as flagellin glycosylation is essential for flagellar assembly in a variety of bacteria (Goon et al., 2003;Schirm et al., 2003;Tabei et al., 2009;Twine et al., 2009;Wu et al., 2011). It is also possible that the presence of sugars may also disguise surface-exposed loop regions of the flagellins from environmental protease cleavage, as N-glycans at the C. jejuni cell surface have been shown to potentially protect proteins from gut proteases (Alemka et al., 2013). It could also be required for favorable interactions with host-cell surfaces, as flagella are essential for bacterial adherence to host cells and therefore the first stages of colonization . However, if flagellin glycosylation is heterologous, the movement of pseudaminic acid around the flagellum may disguise this appendage from the host's immune system during infection or it is also possible that flagellin glycosylation may vary depending on the bacterium's situation. Campylobacter flagellar glycosylation varies due to phase variation of the glycosyltransferase proteins (Maf proteins) (Van Alphen et al., 2008), of which it has several and is capable of glycosylating its flagella with different nonulosonate sugars (Karkyshev et al., 2002). Furthermore, work by Howard et al. (2009) demonstrated that altered glycosylation levels in C. jejuni affect the overall surface charge of the Campylobacter flagellum, and in doing so, alters the behavior of a population (Howard et al., 2009).
In contrast to the variety of sugars decorating the polar flagella of Campylobacter species, Helicobacter pylori glycosylates its polar flagellum solely with Pse5Ac7Ac, similar to A. caviae (Schirm et al., 2003). Little heterogeneity was discovered, with FlaA containing six to seven sugars and FlaB, nine to 10 (Schirm et al., 2003). More recently, glycosylation in the organism, Shewanella oneidensis, has been investigated, with the flagellins being modified with a sugar related to pseudaminic acid, with five sites of modification being confirmed on the dominant flagellin, FlaB, and four sites on FlaA (Bubendorfer et al., 2013;Sun et al., 2013). Heterogeneity between the sites of glycosylation occupied was not reported in these studies (Bubendorfer et al., 2013;Sun et al., 2013). In addition to glycosylation, these studies also identified methylation on both S. oneidensis flagellins; however, sites of methylation did not appear to be essential for either flagellin glycosylation or the production of a functional filament (Bubendorfer et al., 2013;Sun et al., 2013).
Although the biological role of flagellin glycosylation is not well understood, it is clear that bacteria modify their flagella in different ways, and therefore the role of glycosylation may vary in each case.
The previous findings that A. caviae Sch3 glycosylates its flagellins with six to eight pseudaminic acid residues suggest there to be some heterogeneity in the glycosylation process (Tabei et al., 2009). Here, we further investigated the posttranslational modification of the A.
caviae polar flagellins, examining the frequency and location of glycosylation within the protein.

| Bacterial strains, plasmids, and growth conditions
Bacterial strains and plasmids used in this study are listed in Table 1.

| General DNA methods
Where required DNA restriction endonucleases, T4 DNA ligase, and alkaline phosphatase were used as recommended by the suppliers (NEB).

| Generation of pBBR1MCS-5_flaB and site-directed mutants
The flaB gene (918 bp), encoding the A. caviae Sch3 polar flagellin (FlaB) (Gray et al., 2019), was cloned into pBBR1MCS-5 along with a region upstream of the transcription start site to allow expression of flaB from its native promoter (99 bp). Q5 high-fidelity DNA polymerase (NEB) was used to amplify flaB from Sch3 A. caviae genomic DNA with the primers RCL_55 and RCL_56. The PCR product was directly cut with a combination of HindIII and BamHI restriction enzymes before being ligated into HindIII/BamHI cut pBBR1MCS-5 (Kovach et al., 1995). The ligation was transformed into chemically competent E. coli DH5α and minipreps were carried out with resulting transformants to isolate the vector DNA. The presence of flaB was investigated with a BamHI restriction digest and agarose gel electrophoresis (compare with linearized empty pBBR1MCS-5). Likely pBBR1MCS-5_flaB constructs were sent for sequencing using M13 forward and reverse primers to confirm the presence of flaB. Site-directed mutants of FlaB glycosylation sites were generated to determine whether particular glycosylation sites were important for the motility of A. caviae; FlaB site-directed mutants were created via overlap extension PCR (OE-PCR), where serine and threonine residues on the peptide, 146 FQVGADANQ-TIGFSLSQAGGFSISGIAK 173 , were mutated to alanine residues, due to the structural simplicity and nonpolar properties of this amino acid.

| Motility assays
To assess the motility of Aeromonas strains, bacterial colonies were transferred with a sterile toothpick into the center of motility agar plates (1% tryptone, 0.5% NaCl, 0.25% agar). The plates were incubated face up at 25°C for 14-24 h, and motility was assessed by examining the migration of bacteria through the agar from the center toward the periphery of the plate.

| Flagellin purification method
To purify A. caviae polar flagellins, a flagellar shearing method, adapted from Wilhelms et al. (2012), was carried out as described by Lowry et al. (2015). Briefly, Aeromonas strains were grown on large swarm agar plates and flagella were sheared from the cells via the use of a blender for 10 min. Cells were pelleted by centrifugation at 8000g for 30 min and debris removed from the supernatant by further centrifugation at 18,000g for 20 min. Flagella were pelleted T A B L E 1 Strains and plasmids used in this study Strain or plasmid Genotype and use or description Source or reference Escherichia coli strains DH5α F − Phi80dlacZ ΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 Invitrogen hsdR17(rK-mK+) phoA supE44 lambda-thi-1; used for general cloning S17-1λpir hsdR pro recA, RP4-2 in chromosome, Km::Tn7 (Tc::Mu) λpir, Tp r Sm r De Lorenzo et al. (1990) CC118 λpir Δ(ara leu)7697 araD139 ΔlacX74 galE galK phoA20 thi-1 rspE rpoB(Rf r ) Herrero et al. (1990) argE ( 2.6 | Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting SDS-PAGE and immunoblotting of Aeromonas whole-cell preparations were carried out as previously described (Tabei et al., 2009 To be used in conjunction with RCL_55 to form the front section of flaB for overlap extension PCR. Incorporates a T155A sitedirected mutation into FlaB.
To be used in conjunction with RCL_56 to form the back section of flaB for overlap extension PCR. Incorporates a T155A sitedirected mutation into FlaB.
B S159/161A (R) 5′-GGCTTGAGCCAAGGCAAACCCAA-3′ (reverse) To be used in conjunction with RCL_55 to form the front section of flaB for overlap extension PCR. Incorporates S159/161A site-directed mutations into FlaB.
To be used in conjunction with RCL_56 to form the back section of Sch3N flab for overlap extension PCR. Incorporates S159/ 161A site-directed mutations into FlaB.
B S167/169A(R) 5′-CAATCCCAGCAATGGCGAACCCTC-3′ (reverse) To be used in conjunction with RCL_55 to form the front section of flaB for overlap extension PCR. Incorporates S167/169A site-directed mutations into FlaB.
To be used in conjunction with RCL_56 to form the back section of flaB for overlap extension PCR. Incorporates S167/169A sitedirected mutations into FlaB.
To be used in conjunction with RCL_55 to form the front section of flaB for overlap extension PCR. Incorporate an I168A sitedirected mutation into FlaB.
To be used in conjunction with RCL_56 to form the back section of flaB for overlap extension PCR. Incorporates an I168A sitedirected mutation into FlaB.
To be used in conjunction with RCL_55 to form the front section of flaB for overlap extension PCR. Incorporates an L160A sitedirected mutation into FlaB.
To be used in conjunction with RCL_56 to form the back section of flaB for overlap extension PCR. Incorporates an L160A sitedirected mutation into FlaB.
Note: Restriction sites or changed codons for site directed mutagenesis are indicated in bold type. membrane. Following the transfer, membranes were blocked with 5% (w/v) powdered skimmed milk. For identification of flagellin, membranes were probed with a polyclonal rabbit anti-polar flagellin antibody (1:10,000) that only recognizes glycosylated flagellin or a rat anti-polar flagellin antibody (1:1,000) that recognizes both glycosylated and unglycosylated flagellin . A goat antirabbit or goat anti-rat horseradish peroxidase-conjugated secondary antibody (1:5000) was used in combination with the ECL detection system (GE Healthcare) before being exposed to X-ray film and developed using a Compact X4 automatic film processor (Xograph Healthcare).

| Statistical analysis
The differences between the wild-type and mutant strains and the mutant strains versus the complemented strains were analyzed using GraphPad Prism 5.0 (GraphPad Software). Data are given as means ± the standard error of the mean (SEM). Statistical significance was compared to the wild-type by t test or one-way analysis of variance (Tukey's multiple comparisons test).

| Trypsin digest of flagellin
In-gel trypsin digestion was performed as previously described (Couto et al., 2011;Shevchenko et al., 2006).  water, respectively, as demonstrated previously Thibault et al., 2001). Although many glycopeptides could be manually visualized within the spectra (recorded between 60 and 100 min retention times), many difficulties were faced when trying to identify them. Due to the lack of lysine and arginine residues, tryptic peptides within this D2/D3 region are large; therefore, there are many possible sites of glycosylation on each tryptic peptide.
Other enzymes such as LysC, GluC, and chymotrypsin did not cut frequently enough on their own, but when used in combination,

| The Aeromonas caviae flagellin is also methylated
In other bacteria such as Shewanella, lysine residues of flagella have been demonstrated to be methylated (Sun et al., 2013). Therefore, methylation of lysine residues was also investigated in A. caviae.
When methylation of lysine residues (methyl/dimethyl/trimethyl) was introduced into the EasyProt search parameters, a variety of methylated peptides were discovered. The FlaA peptide, 108 DREALQKEVDQLGAEINR 125 , was suggested to be present in numerous forms in the wild-type A. caviae flagellin sample, as evidence for dimethylation and trimethylation on the central lysine 114 was discovered, along with the unmodified peptide ( Figure 2a).

| Removal of glycosylation sites affects motility
As the FlaB peptide, 146 FQVGADANQTIGFSLSQAGGFSISGIAK 173 , was identified to be glycosylated (potentially with either one or two pseudaminic acid residues), and the site of glycosylation appears to vary, FlaB was selected for site-directed mutagenesis studies to investigate whether any specific glycoforms of this peptide are particularly required for the motility of A. caviae. These studies were carried out in an A. caviae flaA flaB double mutant background strain (Rabaan et al., 2001) into which the wild-type copy or site-directed mutants were introduced. A single mutation was made in T155, double mutations in S159/161 or S167/169, and quadruple mutations in S159/161/167/169. The wild-type copy and site-directed flaB mutants were cloned separately into the broad host range vector pBBR1MCS-5.
The plasmid, pBBR1MCS-5_flaB, and the vectors containing desired site-directed mutations (T155A, S159/161A, S167/169A, or S159/161/167/169A) were conjugated into an A. caviae flaAB mutant (Rabaan et al., 2001). Swimming motility assays were carried out on large swimming motility plates, allowing an A. caviae flaAB mutant expressing the desired FlaB site-directed mutant to be analyzed alongside the flaAB mutant alone, the mutant containing the empty vector, and the mutant containing wild-type flaB.
As expected, the A. caviae flaAB mutant alone was nonmotile as was the version complemented with the vector pBBR1MCS-5 only. When the wild-type version of the FlaB was introduced into the mutant motility was restored. The flaB(T155A) construct was able to complement the nonmotile phenotype of the A. caviae flaAB mutant and was found to swim significantly more (18% more) than the mutant expressing wild-type FlaB (Figure 3a). Expression of the pBBR1MCS-5 constructs containing the double mutations, flaB(S159/161A) and flaB(S167/169A), were both found to restore motility in the flaAB mutant; however, the motility was significantly impaired (Figure 3b). These strains displayed a reduction in motility of 26% (S159/161) and 39%  . The radius of each motility halo was measured after 24 h and average measurements are presented here (n = 6) plus or minus the standard error of the mean. p < 0.0001 was generated when a paired t test was carried out on the data sets.
However, although both FlaB(S159/161A) and FlaB(S167/169A) can produce a functional flagellum, they are observed as thinner bands on the Western blots compared to wild-type FlaB; this is potentially due to only possessing the lower levels of flagellin glycosylation (i.e., restricted to possessing only six to seven pseudaminic acid residues, whereas wild-type flagellin is free to occupy more sites). Additionally, the levels of both FlaB(S159/ 161A) and FlaB(S167/169A) visually appear to be lower than the levels of wild-type FlaB or FlaB(T155A). Furthermore, a noticeable size shift can be observed when wild-type FlaB samples, FlaB

| Mutation of D2/D3 domain hydrophobic amino acids affects motility
For N-linked glycosylation, there is a known consensus sequence and there is no known consensus sequence for O-linked flagellin glycosylation (Nothaft & Szymanski, 2010). Several studies have recognized that the specific sites of glycosylation appear to occur in highly hydrophobic regions of the protein, suggesting there to be some selectivity to the glycosylation process (Schirm et al., 2003;Thibault et al., 2001). The Kyte and Doolittle hydrophobicity plot in However, one glycopeptide was confidently identified from FlaB: 146 FQVGADANQTIGFSLSQAGGFSISGIAK 173 . It was observed that this peptide can be glycosylated with either one or two pseudaminic acid residues and that the site of modification can vary on the peptide. Glycosylation of the two central serine residues (serine 159 and 161) was detected most frequently. This peptide was never found in its unmodified form in wild-type flagellin samples, suggesting that the glycosylation of this D2/D3 domain peptide, in particular, is important for the formation of a functional flagellar filament. This was supported by the site-directed mutagenesis studies, where the double mutations at S159/161A and S167/169A, caused a significant decrease in A. caviae motility and less flagellin protein being present.
This suggests that these mutant flagellins may fold in a way that makes them more readily degraded, maybe due to their less efficient polymerization, or they are less stable compared to the wild-type flagellins. Mass spectrometric analysis of the mutated flagellin, FlaB (S159/161A), uncovered that the mutated FlaB [146-173, S159/ 161A] peptide was still glycosylated, but only ever with a single sugar. Here, pseudaminic acid occupied any of the remaining serine and threonine sites, with serine 167 being identified as the most frequent. It is, therefore, possible that the decreased A. caviae motility observed when only this mutated flagellin is present, maybe due to the flagella formed having a decreased hydrophilicity, and therefore impaired ability to move through an aqueous environment.
In addition, it has previously been noted that unglycosylated flagellin is recognized less by the chaperone, FlaJ .
Therefore, reduced flagellin glycosylation may lead to a reduced recognition of the flagellins by FlaJ, leading to decreased flagellin export and therefore filament polymerization. However, there is also less protein present within the bacterial cell, which may affect flagellar assembly and filament length, suggesting that there may be protein degradation in the less glycosylated versions. Site-directed mutagenesis studies of the 19 sites of glycosylation in C. jejuni have also shown specific glycosylation sites to have an impact on motility, where the individual mutation of three serine residues in particular (in C. jejuni 81-176) caused the formation of truncated flagellar filaments and therefore reducing bacterial motility (Ewing et al., 2009). Although single site-specific mutants of serine had little effect on motility, this was only seen when one or more residues were mutated. Conversely, mutation of the single threonine (T155A) caused an increase in motility, this could be due to optimal folding or increased flagellin stability. Impaired motility is also observed when the mutated flagellin, FlaB(S167/169A), is the only flagellin present.
The two mutated residues here were never found to be comodified in wild-type flagellin samples, and therefore it is likely that the serine residues, 159 and 161, are still the predominantly modified residues on the FlaB peptide [146-173, S167/169A]. As impaired motility is also seen with the comutation of these residues, it is possible that this phenomenon is not related to flagellin glycosylation, but occurs due to the increased hydrophobicity of the peptide (from mutating serine to alanine and the loss of Pse5Ac7Ac), resulting in an overall increase in the hydrophobicity of the polar flagellum. This may lead to less favorable interactions with the aqueous environment.
When all four serine residues on this peptide (S159, S161, S167, and S169) were mutated, the flagellin was found to be smaller than the wild-type and other site-directed mutant flagellins. Furthermore, the size of this flagellin also demonstrates that when this region of the protein is not glycosylated it cannot be compensated for by glycosylation of the other remaining sites. This further indicates that this region is essential for motility. The mutation of these residues may result in the improper folding of the flagellin, or these flagellins may polymerize differently from the wild-type flagellins and be the reason for the impaired and nonmotile phenotypes visualized here.
Although the D2/D3 region in other bacterial flagellins allows large deletions or additions within this domain with little effect on filament formation (Reid et al., 1999;Szabo et al., 2011).

Previous studies into bacterial O-linked flagellin glycosylation
have suggested that local regions of hydrophobicity may play a role in the glycosyltransferase selection of these residues (Schirm et al., 2003;Thibault et al., 2001). In particular, Thibault et al. (2001) identified hydrophobic amino acid sequences preceding their identified sites of flagellin modification in C. jejuni. Hydrophobic amino acids are also present in the D2/D3 domain of A. caviae flagellins, preceding the serine residues 159 and 161, and the serine 167 and 169 residues. However, hydrophobic amino acids did not precede threonine 155, which may show that this residue is not the desired target for glycosylation and be why mutation of this residue did not impair motility. Furthermore, the double serine residues on this peptide are both separated by hydrophobic amino acids, which led to the selection of leucine 160 and isoleucine 168 for further sitedirected mutagenesis studies (L160A and I168A). Both mutations resulted in severely reduced A. caviae motility and only low levels of these proteins were detected in whole-cell and supernatant samples.
It is, therefore, possible that these mutated proteins are readily degraded, or less stable than their wild-type counterparts. It is also possible that the mutations present may affect the ability of these flagellins to be secreted or affect the folding of FlaB, having an effect on flagellar assembly.
Although there is no consensus sequence for O-linked glycosylation, it is tempting to speculate that these local regions of hydrophobicity within the D2/D3 domains of the flagellins, may guide the glycosyltransferase Maf1, to the preferred residues for modification, allowing more frequent glycosylation of these residues.
As this is not a fixed consensus sequence, it indicates the modification process to be flexible, permitting other residues to also be modified, although less commonly, and resulting in a heterogeneous flagellin glycosylation pattern that we have observed.
Methylation was also detected on lysine residues on both A.
caviae flagellins (FlaA/B). This modification did not appear to be essential for flagellar formation and motility. As the methylated peptides were also identified in their unmodified forms. This is not the first time that methylation has been identified on glycosylated bacterial flagellins, as S. oneidensis flagellins have also been found to be methylated on at least five lysine residues on the dominant flagellin, FlaB (Bubendorfer et al., 2013;Sun et al., 2013). The role of flagellin methylation in S. oneidensis is unclear; however, mutation of the putative methyltransferase did not affect bacterial motility (Sun et al., 2013). Interestingly, the recently published structure of the Maf glycosyltransferase suggests one of its three domains shares similarities with a methyl transferase (Sulzenbacher et al., 2018).  (Takeuchi et al., 2003), and although this is not essential for swimming and swarming motility, glycosylation mutants have a decreased ability to adhere to surfaces and cause disease in the plant (Taguchi et al., 2006).
Site-directed mutagenesis studies have revealed that sites of modification located on the surface of the flagellum are more essential for bacterial virulence (Taguchi et al., 2006). However, the individual site-directed mutagenesis of the known sites of modification were all found to impair motility, compared to when optimum levels of flagellin glycosylation are present (Taguchi et al., 2010). Glycosylated flagellins have been found to polymerize into a more stable flagellum, being more heat resistant than the unglycosylated equivalent (Taguchi et al., 2009).

CONFLICT OF INTEREST
None declared.

DATA AVAILABILITY STATEMENT
All data are provided within this manuscript. Data for the manual analysis of the flagellin glycopeptides are available in the Zenodo repository at https://doi.org/10.5281/zenodo.6796498.

ETHICS STATEMENT
None declared.